Mesangial cell-derived receptors for advanced glycosylation endproducts and uses thereof

ABSTRACT

The present invention relates to a method and associated agents for measuring the presence and amount of advanced glycosylation endproducts in cells and fluids. The methods take advantage of the existence of receptors and receptor complexes for AGEs and include receptor-containing ligands comprising whole mesangial and other cells, mesangial cellular fragments and protein extracts therefrom. Competitive assays, sandwich assays and assays involving AGE antisera are disclosed. Numerous diagnostic applications are defined and test kits are also contemplated.

This invention was made with partial assistance from grant Nos. AG 8245and DK 19655 from the National Institutes of Health. The government mayhave certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Application is a continuation-in-part of application Ser.No. 453,958, filed Dec. 20, 1989, which is in turn, a Division ofapplication Ser. No. 091,534, filed Sep. 3, 1987, now U.S. Pat. No.4,900,747, issued Feb. 13, 1990, which is in turn, acontinuation-in-part of application Ser. No. 907,747, filed Sep. 12,1986, now abandoned; all of the above preceding applications by HelenVlassara, Michael Brownlee and Anthony Cerami, said Ser. No. 907,747, inturn, a continuation-in-part of application Ser. No. 798,032, filed Nov.14, 1985, by Anthony Cerami, Peter Ulrich and Michael Brownlee, now U.S.Pat. No. 4,758,583, which is, in turn, a continuation-in-part ofapplication Ser. No. 590,820, now U.S. Pat. No. 4,665,192, filed Mar.19, 1984 by Anthony Cerami alone.

Priority under 35 U.S.C. §120 is claimed as to all of the above earlierfiled Applications, and the disclosures thereof are incorporated hereinby reference.

RELATED PUBLICATIONS

The Applicants are co-authors of the following articles directed to thesubject matter of the present invention: "FUNCTION OF MACROPHAGERECEPTOR FOR NONENZYMATICALLY GLYCOSYLATED PROTEINS IS MODULATED BYINSULIN LEVELS", Vlassara, Brownlee and Cerami, DIABETES (1986), Vol. 35Supp. 1, Page 13a; "ACCUMULATION OF DIABETIC RAT PERIPHERAL NERVE MYELINBY MACROPHAGES INCREASES WITH THE PRESENCE OF ADVANCED GLYCOSYLATIONENDPRODUCTS", Vlassara, H., Brownlee, M., and Cerami, A. J. EXP. MED.(1984), Vol. 160, pp. 197-207; "RECOGNITION AND UPTAKE OF HUMAN DIABETICPERIPHERAL NERVE MYELIN BY MACROPHAGES", Vlassara, H., Brownlee, M., andCerami, A. DIABETES (1985), Vol. 34, No. 6, pp. 553-557;"HIGH-AFFINITY-RECEPTOR-MEDIATED UPTAKE AND DEGRADATION OFGLUCOSE-MODIFIED PROTEINS: A POTENTIAL MECHANISM FOR THE REMOVAL OFSENESCENT MACROMOLECULES", Vlassara H., Brownlee, M., and Cerami, A.,PROC. NATL. ACAD. SCI. U.S.A. (September 1985), Vol. 82, pp. 5588-5592;"NOVEL MACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS IS DISTINCTFROM PREVIOUSLY DESCRIBED SCAVENGER RECEPTORS", Vlassara, H., Brownlee,M., and Cerami, A. JOUR. EXP. MED. (1986), Vol. 164, pp. 1301-1309;"ROLE OF NONENZYMATIC GLYCOSYLATION IN ATHEROGENESIS", Cerami, A.,Vlassara, H., and Brownlee, M., JOURNAL OF CELLULAR BIOCHEMISTRY (1986),Vol. 30, pp. 111-120; "CHARACTERIZATION OF A SOLUBILIZED CELL SURFACEBINDING PROTEIN ON MACROPHAGES SPECIFIC FOR PROTEINS MODIFIEDNONENZYMATICALLY BY ADVANCED GLYCOSYLATION END PRODUCTS", Radoff, S.,Vlassara, H. and Cerami, A., ARCH. BIOCHEM. BIOPHYS (1988), Vol. 263,No. 2, pp. 418-423; "ISOLATION OF A SURFACE BINDING PROTEIN SPECIFIC FORADVANCED GLYCOSYLATION ENDPRODUCTS FROM THE MURINE MACROPHAGE-DERIVEDCELL LINE RAW 264.7", Radoff, S., Vlassara, H., and Cerami, A.,DIABETES, (1990), Vol. 39, pp. 1510-1518; "TWO NOVEL RAT LIVER MEMBRANEPROTEINS THAT BIND ADVANCED GLYCOSYLATION ENDPRODUCTS: RELATIONSHIP TOMACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS", Yang, Z., Makita,Z., Horii, Y., Brunelle, S., Cerami, A., Sehajpal, P., Suthanthiran, M.and Vlassara, H., J. EXP. MED., (In Press). All of the foregoingpublications and all other references cited herein are incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the nonenzymaticglycosylation of proteins, and particularly to the discovery of bindingpartners to advanced glycosylation endproducts such as AGE receptors,that may serve in the diagnosis and treatment of conditions in which thepresence or activity of such advanced glycosylation endproducts may beimplicated.

Glucose and other reducing sugars react non-enzymatically with the aminogroups of proteins in a concentration-dependent manner. Over time, theseinitial Amadori adducts undergo further rearrangements, dehydrations andcross-linking with other proteins to accumulate as a family of complexstructures which are referred to as Advanced Glycosylation Endproducts(AGEs). Although this chemistry has been studied by food chemists formany years, it was only in the past decade that the presence of AGEs inliving tissue has been established. The excessive deposition of theseproducts on structural proteins as a function of age and elevatedglucose concentration, taken together with evidence of effectiveprevention of tissue pathology by an AGE inhibitor, aminoguanidine, haslent support to the hypothesis that the formation of AGEs plays a rolein the long term complications of aging and diabetes.

Since the amount of AGEs found in human tissues is less than could bepredicted from protein/glucose incubation studies in vitro, theapplicants herein proposed several years ago that there might be normalmechanisms to remove those long-lived proteins which had accumulatedAGEs in vivo. Particularly, and as set forth initially in Parentapplication Ser. No. 907,747, now abandoned and the above-referencedapplications that have followed, monocytes/macrophages and endothelialcells were found to display high affinity surface binding activityspecific for AGE moieties independent of the protein which wasAGE-modified. This AGE-receptor was shown to differ from other knownscavenger receptors on these cells.

In addition, an endogenous means for the in vivo elimination or removalof advanced glycosylation endproducts was set forth, and correspondingdiagnostic applications involving the receptors and including a specificreceptor assay were also proposed.

Following this determination, the applicants herein have sought tofurther investigate the identity and role of advanced glycosylationendproduct receptors and possible binding partners, and any consequentdiagnostic and therapeuptic implications of these investigations, and itis toward this end that the present invention is directed.

The AGE-specific receptor system now includes a variety of tissues andcell types in addition to monocyte/macrophages for whichreceptor-mediated AGE-protein internalization and digestion was firstdescribed. Endothelial, mesangial cells and fibroblasts have since beenshown to specifically bind AGE-modified protein. In macrophages,AGE-protein uptake is accompanied by the release of a variety of potentcytokines and growth factors, which may coordinate processes of normaltissue remodeling. The other cell types do not bind the model compoundAGE, FFI, nor are they known to release cytokines and growth factors inresponse to AGE-ligand binding, but each cell type does display distinctfunctional responses. For example, endothelial cells exhibit enhancedsurface procoagulant activity and permeability; and mesangial cellsdisplay enhanced matrix protein synthesis; while human fibroblastsincrease proliferation upon exposure to AGEs.

SUMMARY OF THE INVENTION

In accordance with the present invention, a substantially purifiedreceptor as defined herein is disclosed that is derived from mammalianmesangial cells (MCs) that recognizes and binds advanced glycosylationendproducts. The receptor possesses the following characteristics:

A. It recognizes and binds with the ligands AGE-BSA, AGE-RNAse andAGE-collagen I in a saturable fashion, having a binding affinity of2.0±0.4×10⁶ M⁻¹ (500 nM);

B. It recognizes and binds to AGE-BSA which has been reduced with NaBH₄;

C. It does not recognize and bind with the ligand FFI-BSA, unmodifiedBSA, RNAse or collagen I in a solid phase ligand blotting assay; and

D. It comprises one or more of at least three proteins, the first ofsaid proteins having a molecular mass of about 50 kD, the second of saidproteins having a molecular mass of about 40 kD and the third of saidproteins having a molecular mass of about 30-35 kD, as determined bytheir migration on SDS-PAGE.

The present invention also includes various diagnostic and therapeuticutilities predicated on the identification and activities of the MCreceptor for AGEs. Diagnostic utilities include assays such asimmunoassays with labeled receptors, antibodies, ligands and bindingpartners, receptor assays, and screening assays to evaluate new drugs bytheir ability to promote or inhibit production or activity, as desired.The above assays can be used to detect the presence or activity ofinvasive stimuli, pathology or injury, the presence or absence of whichmay affect the structure or function of specific organs.

Therapeutic compositions comprising effective amounts of AGE receptorantagonists, agonists, antibodies or like drugs, etc., andpharmaceutically acceptable carriers are also contemplated. Suchcompositions can be prepared for a variety of protocols, including whereappropriate, oral and parenteral administration. Exact dosage and dosingschedules are determined by the skilled physician.

The invention also includes a method for the measurement of proteinaging both in plants and in animals, by assaying the presence, amount,location and effect of such advanced glycosylation endproducts. Assaysof plant matter and animal food samples will be able, for example, toassess food spoilage and the degradation of other protein material soaffected, while the assays of animals, including body fluids such asblood, plasma and urine, tissue samples, and biomolecules such as DNA,that are capable of undergoing advanced glycosylation, will assist inthe detection of pathology or other systemic dysfunction.

Specifically, the methods comprise the performance of several assayprotocols, involving the analyte, a ligand and one or more bindingpartners to the advanced glycosylation endproducts of interest. Thebinding partners may be selected from the group consisting of cellshaving receptors for advanced glycosylation endproducts, cell componentshaving receptors for advanced glycosylation endproducts, cell proteinscomprising receptors for advanced glycosylation endproducts, andantibodies to the advanced glycosylation endproducts, the receptors,cell components or cell proteins.

The preferred cells having receptors which are useful herein comprisemammalian mesangial cells; the preferred cellular components comprisecell membranes, and the cell proteins are derived from cell membranesand are selected from the group consisting of a 50 kD protein derivedfrom MC membranes, a 40 kD protein derived from MC membranes, and a30-35 kD protein derived from MC membranes, as well as mixtures thereof,having the reactivity which is described herein.

The ligands useful in the present invention are generally AGEderivatives that bind to AGE binding partners. These ligands may bedetected either singly and directly, or in combination with a seconddetecting partner such as avidin. Suitable ligands are selected from thereaction products of reducing sugars, such as glucose andglucose-6-phosphate (G6P), fructose and ribose. These sugars arereactive with peptides, proteins and other biochemicals such as BSA,avidin, biotin, and enzymes such as alkaline phosphatase.

Included in this invention is the discovery and preparation of enzymesand other carriers that undergo advanced glycosylation and that mayserve as labelled ligands in the assays of the present invention. Othersuitable ligands may include synthetic AGEs or the reaction of thesugars directly with carriers capable of undergoing advancedglycosylation. Carriers not so capable may have a synthetic AGE coupledto them. Suitable carriers may comprise a material selected fromcarbohydrates, proteins, synthetic polypeptides, lipids, biocompatiblenatural and synthetic resins, antigens and mixtures thereof.

Whole cell-based assays and standard assays based on either cellcomponents or the cell proteins themselves and employing extracts may beused. The "whole cell" can be intact mesangial cells and/or other cellsbound to ligands for the AGE receptors, such as labelled whole cells.Each assay is capable of being based on enzyme linked and/orradiolabeled AGEs and their binding partners, including the AGEreceptors disclosed herein.

Assay Protocols

The broad format of the assay protocols possible with the presentinvention extends to assays wherein no label is needed for AGEdetection. For example, one of the formats contemplates the use of abound protein-specific AGE receptor. The analyte suspected of containingthe advanced glycosylation endproducts under examination would need onlyto be added to the receptor. The bound analyte could then be easilydetected by a change in the property of the binding partner, such as bychanges in the receptor.

The assays of the invention may follow formats wherein either the ligandor the binding partner, be it a receptor or an antibody, are bound.Likewise, the assays include the use of labels which may be selectedfrom radioactive elements, enzymes and chemicals that fluoresce.

The present method has particular therapeutic relevance as it affords ameans for the detection and evaluation of the condition of a broadspectrum of organ systems. The Maillard process acutely affects severalof the significant protein masses in the body, among them collagen,elastin, lens proteins, and the kidney glomerular basement membranes.These proteins deteriorate both with age (hence the application of theterm "protein aging") and as a result of prolonged exposure to bloodsugar and AGE formation, the latter in turn frequently due to pathology.

In this manner, the location and relative concentrations of advancedglycosylation endproducts in the body can be identified. This techniqueis particularly useful in identifying undesireable concentrations ofadvanced glycosylation endproducts, such as in atheromatous plaques. Insuch manner, the location of the systemic malfunction can be identified.

Accordingly, it is a principal object of the present invention toprovide a method for measuring advanced glycosylation endproducts thatis rapid and reliable.

It is a further object of the present invention to provide a method asaforesaid which is characterized by the discovery and use of the bindingaffinity of receptors for said advanced glycosylation endproducts.

It is a yet further object of the present invention to provide an assayfor the measurement of advanced glycosylation endproducts that iscapable of a broad range of alternative protocols.

It is a yet further object of the present invention to provide an assayas aforesaid that is capable of performance without radioactive labelsand that may be performed in an automated fashion.

Other objects and advantages will become apparent to those skilled inthe art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described in detail in connection with the accompanyingdrawings, as set forth below.

FIG. 1 is a graph depicting the relative binding and uptake of red bloodcells modified with various agents, and illustrating a primary aspect ofthe present invention.

FIG. 2 is a graph illustrating an assay in accordance with the presentinvention by the competitive inhibition in red blood cell binding causedby the introduction into a sample of an agent capable of stimulating redblood cells to increase their activity of recognition and removal ofadvanced glycosylation endproducts.

FIG. 3 is a bar graph illustrating the comparative uptake anddegradation of advanced glycosylation endproducts by mouse macrophagesexposed to various stimulator compounds.

FIG. 4 is a bar graph illustrating data similar to that set forth inFIG. 3, with respect to one day old human monocytes.

FIG. 5--Binding of ¹²⁵ -AGE-BSA to human (A) and rat (B) MC membranes.10 μg of solubilized membrane protein from human and rat mesangial cellswere dot-blotted onto nitrocellulose filters, which were then incubatedwith various concentrations of ¹²⁵ I-AGE-BSA in the presence and absenceof 100-fold excess unlabeled AGE-BSA. Specific binding was obtained bysubtracting the nonspecific binding from the total binding. The insetshows a Scatchard Plot for the specific binding (B=pmoles/ng membraneprotein, F=nM).

FIG. 6--Competitive inhibition of ¹²⁵ I-AGE-BSA binding to human (A) andrat (B) MC membranes. 10 μg of solubilized membrane protein from humanand rat MCs were dot-blotted onto nitrocellulose filters. The filterswere incubated with 50 nM ¹²⁵ I-AGE-BSA for 2 hours at 4° C. Competitionexperiments were performed in parallel experiments in which theradioligand was incubated with 100-molar excess of an unlabeled protein.Data shown are the average of duplicate determinations, and areexpressed as the % maximal binding. Maximal binding was defined as theamount of ¹²⁵ I-AGE-BSA bound in the presence of 100-molar excess coldBSA. Competitors used: (a) BSA, (b) AGE-BSA, (c) NaBH₄ -reduced AGE-BSA,(d) FFI-BSA, (e) AGE-RNAse, (f) RNAse, (g) Collagen I, (h) AGE-collagenI.

FIG. 7--A) Uptake and degradation of ¹²⁵ I -AGE-BSA by rat MCs. MCs wereincubated with various concentrations of ¹²⁵ I-AGE-BSA for 4 hours at37° C. The amount of cell-associated ¹²⁵ I-AGE-BSA (uptake), and theamount of trichloroacetic acid-soluble counts in the medium(degradation) were determined in triplicate wells. B) Accumulation of¹²⁵ I-AGE-BSA versus time. MCs in each well were incubated with 20 μg of¹²⁵ I-AGE-BSA at 37° C. and specific cell-associated radioactivity wasdetermined at various time intervals. Cellular accumulation ofradioactivity is expressed as the % of the maximal accumulation of ¹²⁵I-AGE-BSA.

FIG. 8--Ligand blot analysis of enriched human MC membranes. 10 μg ofsolubilized membrane protein were electrophoresed on a nonreducingSDS/polyacrylamide gel (10%). The proteins on the gel wereelectroblotted onto nitrocellulose membrane and probed with ¹²⁵I-AGE-BSA in the presence of 100-fold excess of either BSA (lane a) orAGE-BSA (lane b). The analysis presented is one of four identicalexperiments.

FIG. 9--Effects of AGE-matrices on [³ H]thymidine incorporation by MCs.Rat MCs were plated onto various matrices (10 μg/ml), as described: (a)Fibronectin, (b) AGE-Fibronectin, Fibronectin, (c) Collagen I, (d)AGE-Collagen I, (e) Laminin, (f) AGE-Laminin. The results are expressedas the means±SEM of 6 experiments and are expressed as the % of [³H]thymidine incorporated relative to the control value, with controlrepresenting [³ H]thymidine incorporated by cells plated on plastic.

FIG. 10--Effect of AGE-matrices on fibronectin synthesis by MCs. HumanMCs were plated onto either unmodified or AGE-modified matrices andlabeled with 35S-methionine and cysteine, as described. The amount offibronectin released into the medium (A), and incorporated into thematrices (B), was determined by immunoprecipitation. The fibronectinbands on the gel were excised and counted for radioactivity. The valuesshown are expressed as the % increase in fibronectin produced by cellsplated on the AGE-matrices relative to that produced by cells plated oncontrol unmodified matrices. The values show cpm/well and represent themeans±SEM from 4 experiments. a) Fibronectin, b) Polylysine, c) CollagenI.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g. Maniatis, Fritsch & Sambrook,"Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: APractical Approach," Volumes I and II (D. N. Glover ed. 1985);"Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic AcidHybridization" (B. D. Hames & S. J. Higgins eds. 1985); "TranscriptionAnd Translation" (B. D. Hames & S. J. Higgins eds. 1984); "Animal CellCulture" (R. I. Freshney ed. 1986); "Immobilized Cells And Enzymes" (IRLPress, 1986); B. Perbal, "A Practical Guide To Molecular Cloning"(1984).

As used herein, the term "AGE-" refers to the advanced glycosylationendproducts of the compound to which it relates. This compound istypically a protein having terminal amino groups which are reactive withreducing sugars. Examples include bovine endothelial ribonuclease(RNAse), human serum albumin (HSA), bovine serum albumin (BSA), collagentype (I) and other proteins.

As used throughout the present application, the term "receptor complex"includes both the singular and plural and contemplates the existence ofa one or more receptor structures which are in turn comprised of theindividual proteins defined herein.

The abbreviation "FFI" refers to the model AGE2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole, which can be reacted with aprotein, e.g., BSA, by adding 100 mM carbodiimide to form the adductFFI-BSA.

As noted above, the preferred cells containing AGE receptors are MCs.Mesangial cells are contained in the mammalian kidneys and function inconjunction with the glomeruli to regulate the glomerular filtrationrate, thus affecting glomerular flow. Increases in the mesangial matrixhave been shown to decrease the filtering surface of the glomerulus, andthus impinge upon the glomerular capillary vasculature. This is due tothe accumulation of normal matrix proteins, e.g., collagen type IV, typeV, laminin and fibronectin.

Mesangial cells have been evaluated as described herein, and found tocontain AGE receptors. The specific findings that follow derive from theexperiments the procedures of which are set forth in detail in Example3, presented below. When AGE-modified proteins accumulate in themesangial matrix and bind to these receptors, MC proliferation,synthesis, metabolism and physiology are modified. For example, theproliferation of MCs is reduced when AGEs have reacted With MC receptorsas compared to MCs Which have not been exposed to AGEs.

Additionally, when AGE-modified proteins are bound to MCs, an increasein fibronectin production is observed, which in turn causes an adversebuild-up of the mesangial matrix.

The MC receptors which recognize AGEs are present on the cell membranes,and demonstrate binding to AGE-modified proteins in a saturable fashionwith a binding affinity of about 2.0±04×10⁶ M⁻¹ (kD=500 nM). Thisbinding is specific for AGE-modified proteins; non-AGE modified proteinsdo not compete for receptor recognition in binding assays.

The MC receptors are comprised of at least 3 distinct proteins, 50 kD,40 kD and 30-35 kD. This was demonstrated using a ligand blotting assayof MC membrane extracts, the procedure of which is described below. Theresults are shown in FIG. 8.

The MC AGE receptor was further characterized with respect to itsbinding affinity, using a series of assays. It was determined that theMC membrane AGE receptors bind AGEs in a saturable fashion. (See FIG.5.) When MC membrane extracts were exposed to increasing levels of AGEs,specific binding plateaued, even as AGE levels were increased.Half-maximal binding occurred at about a 150nM concentration of AGE-BSA.This was consistent between rat and human MC membrane extracts. Thenumber of AGE molecules bound per cell was in the range of 3.0±0.25×10⁵molecules per cell. The binding affinity constant was 2.0±0.40×10⁶ M⁻¹(kD=500 nM).

Similar results were observed when whole MCs were assayed (data notshown).

The MC receptor binding affinity was further evaluated, and it wasdetermined that the MC receptor for AGE-modified proteins is reactivewith AGE-modified proteins and non-reactive with proteins in unmodifiedform. These conclusions were drawn based upon competitive binding assaysrun wherein the MC receptor for AGEs is dot-blotted onto anitrocellulose filter, blocked with BSA, quantitated with labelledligand and then competitive assayed with labelled ligand and the proteinto be evaluated. Competition was evaluated based upon the level ofreactivity, compared to that which was present when no competing proteinwas included.

Specific binding to the receptors was defined as the difference betweentotal binding (radioligand incubated with membrane protein alone) andnon-specific binding (cell incubated with radiolabelled ligand plus 100fold excess of unlabelled ligand).

Scatchard analysis of the data was performed to determine the bindingaffinity constant and the receptor number. See Scatchard, G. Ann. N.Y.Acad. Sci. (1949) 51: 660-72.

The competitive binding assays were run using rat and human MC extracts,with the results shown in FIG. 6. It was confirmed that AGE-modifiedproteins were binding to the receptors. Excess cold AGE-BSA (FIG. 6Ab,6Bb) competed with labelled AGE-BSA. The excess cold AGE-BSAcompetitively inhibited greater than 80% of ¹²⁵ I-AGE-BSA binding to MCmembrane extract, and other AGE-modified proteins, namely AGE-RNAse(FIG. 6Ae, 6Be), and AGE-collagen I (FIG. 6Ah) competed effectively with¹²⁵ I-AGE-BSA.

Unmodified BSA (FIG. 6Aa, 6Ba) did not compete, nor did excessunmodified RNAse or collagen I (FIGS. 6Af, 6Bf and 6Ag).

The AGE-modified protein receptors were further characterized withrespect to binding for AGE-BSA in reduced form. AGE-BSA was reduced withNaBH₄ to glucitolysine and further evaluated. The reduced AGE-BSAeffectively competed with radiolabelled AGE-BSA for binding to MCmembrane extracts (FIGS. 6Ac, 6Bc).

The chemically synthesized model AGE-, FFI was assessed for itscompetitive ability to bind to MC AGE-receptors. The results are shownin FIG. 6Ad and 6Bd. It did not compete with labelled AGE-BSA. The MCreceptor does not recognize FFI-BSA.

The characteristics of the MC AGE-modified protein receptor wereevaluated to determine the number of proteins involved and theirmolecular weight. It was determined that three distinct membraneproteins are present in the receptor complex, 50 kD, 40 kD and 30-35 Kd,using a ligand blot analysis.

The receptor proteins were evaluated using detergent extracts of MCmembranes. FIG. 8 shows three prominent AGE binding proteins. Binding ofradiolabelled AGE to these membrane proteins was specific. Again, excessunlabelled AGE-BSA could inhibit labelled AGE-binding for both rat andhuman MCs, whereas BSA did not inhibit labelled AGE binding.

The effect of AGEs on mesangial cell metabolism was evaluated using anumber of different comparisons. Typically, the analysis addressed aparticular metabolic parameter in AGE-modified MCs, and compared it toMCs unexposed to AGEs. To evaluate the effect of AGEs on MCproliferation, MCs were incubated with and without AGE-modified proteinsin the presence of labelled thymidine. (Thymidine is taken up during DNAsynthesis). The results are shown in FIG. 9. AGE-modified MCs showed adecrease in label uptake over MCs not exposed to AGEs.

MC proliferation was also evaluated in the presence of matrix proteins,fibronectin, collagen I and laminin, comparing MC growth on platescoated with these matrix proteins to that which occurs in the presenceof these matrix proteins in AGE modified form. The results are shown inFIG. 9. Labelled thymidine uptake was used as the measure of MCproliferation. When used in a concentration of 10 mg/ml, the uptake oflabelled thymidine uptake was consistently reduced in the presence ofAGE-modified matrix proteins, approximately to the level ofincorporation in MCs plated on plastic.

In contrast cells grown on collagen I and fibronectin coated plates(non-AGE modified) showed enhanced thymidine uptake; cells grown onnon-AGE modified laminin coated plates showed no stimulated uptake overcontrol values.

The reduced thymidine uptake in the presence of AGE modifiedfibronectin, collagen I and laminin was confirmed with Brd Uincorporation assays used to control for DNA synthesis.

The uptake and degradation of AGEs by MCs was also evaluated using theprocedures of Vlassara, H. et al. Proc. Natl. Acad. Sci. USA (1985);82:5588-92 (modified slightly). MC accumulation of labelled ligand(AGE-BSA) was assessed by incubating cells with various concentrationsof labelled AGE-BSA in the presence or absence of a 100 fold excess ofunlabelled AGE-BSA. The amount of cell-contained label was determined.This is generally indicative of AGE uptake by MCs. The results are shownin FIGS. 7. Uptake data is shown in FIGS. 7A and 7B. MCs continued toaccumulate AGEs beyond the level of AGE-binding to as high as 1.1 μM.

Since the level of AGE accumulation could include both bound AGEs andinternalized AGEs, accumulation at 4° C. was compared to that at 37° C.(At 4° C. AGEs bind to MCs, whereas at 37° C. AGEs are internalized byMCs.) The maximum accumulation of labelled AGE-BSA occurred within 2hours of incubation at 37° C. (FIG. 7B). The amount of label which wascell associated at 37° C. was 2-4 times higher than the amount of labelbound to MCs at 4° C.

Degradation of AGEs was determined by measuring the amount of labelpresent in the aspirated medium.

Concommitant with MC accumulation of labelled AGE-BSA, liganddegradation also increased, as measured by a steady increase intrichloroacetic acid (TCA) soluble radioactivity in the media. Theincrease in AGE-BSA degradation paralleled the increase in MC uptake ofAGE-BSA. (FIG. 7A).

The effect of AGEs on fibronectin production in MCs was also evaluated.Mesangial cells were grown on AGE-modified or unmodified collagen I,fibronectin and polylysine media. The amount of fibronectin releasedinto the medium (or incorporated into the matrix) was determined byimmunoprecipitation with IgG purified anti-human fibronectin antibodies.The results are shown in FIG. 10, showing an increase in the synthesisof fibronectin in the presence of AGE-modified proteins.

The mesangial cells receptors for AGE-modified proteins can be furthercharacterized by comparing said receptors to other known AGE-receptors,such as on macrophage and endothelial cells. AGEs have been shown tobind to specific receptors on murine and human monocyte/macrophages, aswell as on bovine and human endothelial cells. Since AGEs formprogressively over time, the function of AGE receptors is believed toinclude signalling cells to promote turnover of aging tissue. Undernormal conditions, the removal of AGE-modified proteins occurs at a ratewhich keeps up with production, thereby preventing accumulation.

In diabetes, excessive AGE formation in the presence of elevated glucoselevels results in a net increase of AGEs on most structural tissueproteins.

Macrophages are induced to release the cytokines catechin/tumor necrosisfactor (TNF), interleukin-1 (IL-1), platelet derived growth factor(PDGF), and insulin-like growth factor (ILGF). Excessive blood glucoselevels may lead to an exaggerated response, which could contribute tocomplications, e.g., atherosclerotic plaque formation, mesangialexpansion and the like.

Endothelial cell AGE receptors induce several changes in EC functionwhich, are characteristic in diabetes, such as increased EC permeabilityand procoagulant properties.

The macrophage AGE receptors recognize the model AGE, FFI-BSA; incontrast, endothelial cell AGE receptors do not recognize the AGE-FFI-BSA.

The ability of MCs to slowly internalize and degrade AGE-modifiedproteins points to a contributing role for MCs in the turnover andremodeling of senescent AGE-modified mesangial matrix proteins. Theefficiency with which MCs ingest and degrade AGE-BSA is approximately10% of that which has been reported for the macrophage. While the AGEreceptors on MCs, macrophage cells and endothelial cells all recognizeAGEs to a degree, there are other differences. First, the Kds for thereceptors are different. The Kd for macrophage and endothelial cell AGEreceptors is about 70-100 nM; the Kd for MC AGE receptors is about 500nM.

In addition, the efficiencies at which the AGE receptors from thesethree cell types internalize and degrade AGE-modified proteins aredifferent. Macrophage AGE receptors ingest and degrade AGE-BSA at a muchhigher rate than do endothelial cells or MCs.

Lastly, macrophage AGE receptors recognize the specific adduct FFIO-BSA,whereas endothelial cell and MC AGE receptors do not. These differencesin reactivity can be used to characterize the AGE receptor proteinsderived from their respective cell types.

In accordance with the present invention, methods have been developedfor the measurement of the presence and amount of advanced glycosylationendproducts in both plants and animals, including humans. The methodscomprise assays involving in addition to the analyte, one or morebinding partners of the advanced glycosylation endproducts, and one ormore ligands.

Accordingly, the present assay method broadly comprises the steps of:

A. preparing at least one biological sample suspected of containing saidadvanced glycosylation endproducts;

B. preparing at least one corresponding binding partner directed to saidsamples;

C. placing a detectible label on a material selected from the groupconsisting of said samples, a ligand to said binding partner and saidbinding partner;

D. placing the labeled material from Step C in contact with a materialselected from the group consisting of the material from Step C that isnot labeled; and

E. examining the resulting sample of Step D for the extent of binding ofsaid labeled material to said unlabeled material.

Suitable analytes may be selected from plant matter, blood, plasma,urine, cerebrospinal fluid, lymphatic fluid, and tissue; and thecompounds FFI and AFGP, individually and bound to carrier proteins suchas the protein albumin. The analyte may also comprise a syntheticallyderived advanced glycosylation endproduct which is prepared, forexample, by the reaction of a protein or other macromolecule with asugar such as glucose, glucose-6-phosphate, or others. This reactionproduct could be used alone or could be combined with a carrier in thesame fashion as the FFI-albumin complex.

The carrier may be selected from the group consisting of carbohydrates,proteins, synthetic polypeptides, lipids, biocompatible natural andsynthetic resins, antigens and mixtures thereof.

The present invention is also useful in diagnosing both the degradativeeffects of advanced glycosylation of proteins in plants and the like,and the adverse effects of the buildup of advanced glycosylationendproducts in animals. Such conditions as age- or diabetes-relatedhardening of the arteries, skin wrinkling, arterial blockage, anddiabetic, retinal and renal damage in animals all result from theexcessive buildup or trapping that occurs as advanced glycosylationendproducts increase in quantity. Therefore, the diagnostic method ofthe present invention is used to detect/or avert pathologies caused atleast in part by the accumulation of advanced glycosylation endproductsin the body by monitoring the amount and location of such AGEs.

Likewise, as advanced glycosylation endproducts may be measured by theextent that they bind to receptors on cells from a variety of sources,the assays of the present invention have been designed and may beperformed around this activity. For example, in a typical competitiveassay in accordance with the present invention, the receptor or cellularmaterial bearing the receptor may be combined with the analyte and theligand and the binding activity of either or both the ligand or theanalyte to the receptor may then be measured to determine the extent andpresence of the advanced glycosylation endproduct of interest. In thisway, the differences in affinity between the components of the assayserves to identify the presence and amount of the AGE.

The present invention also relates to a method for detecting thepresence of stimulated, spontaneous, or idiopathic pathological statesin mammals, by measuring the corresponding presence of advancedglycosylation endproducts. More particularly, the activity of AGEs maybe followed directly by assay techniques such as those discussed herein,through the use of an appropriately labeled quantity of at least one ofthe binding partners to AGEs as set forth herein. Alternately, AGEs canbe used to raise binding partners or antagonists that could in turn, belabeled and introduced into a medium to test for the presence and amountof AGEs therein, and to thereby assess the state of the host from whichthe medium was drawn.

Thus, both AGE receptors and any binding partners thereto that may beprepared, are capable of use in connection with various diagnostictechniques, including immunoassays, such as a radioimmunoassay, usingfor example, a receptor or other ligand to an AGE that may either beunlabeled or if labeled, then by either radioactive addition, reductionwith sodium borohydride, or radioiodination.

In an immunoassay, a control quantity of a binding partner to advancedglycosylation endproducts may be prepared and optionally labeled, suchas with an enzyme, a compound that fluoresces and/or a radioactiveelement, and may then be introduced into a tissue or fluid sample of amammal believed to be undergoing invasion. After the labeled material orits binding partner(s) has had an opportunity to react with sites withinthe sample, the resulting mass may be examined by known techniques,which may vary with the nature of the label attached.

The presence of AGE activity in animals and plants can be ascertained ingeneral by immunological procedures are which utilize either a bindingpartner to the advanced glycosylation endproduct or a ligand thereto,optionally labeled with a detectable label, and further optionallyincluding an antibody Ab₁ labeled with a detectable label, an antibodyAb₂ labeled with a detectable label, or a chemical conjugate with abinding partner to the advanced glycosylation endproduct labeled with adetectable label. The procedures may be summarized by the followingequations wherein the asterisk indicates that the particle is labeled,and "R" in this instance stands for all binding partners of advancedglycosylation endproduct(s) under examination although particularreference is to be made to the receptor complex of the presentinvention:

A. BP+Ab=BP*Ab₁

B. BP+Ab*=BPAb₁ *

C. BP+Ab₁ +Ab₂ *=BPAb₁ Ab₂ *

D. Carrier *BP+Ab₁ =Carrier*BPAb₁

These general procedures and their application are all familiar to thoseskilled in the art and are presented herein as illustrative and notrestrictive of procedures that may be utilized within the scope of thepresent invention. The "competitive" procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Optional procedureC, the "sandwich" procedure, is described in U.S. Pat. Nos. RE 31,006and 4,016,043, while optional procedure D is known as the "doubleantibody", or "DASP" procedure.

A further alternate diagnostic procedure employs multiple labeledcompounds in a single solution for simultaneous radioimmune assay. Inthis procedure disclosed in U.S. Pat. No. 4,762,028 to Olson, acomposition may be prepared with two or more analytes in a coordinatedcompound having the formula: radioisotope-chelator-analyte.

In each instance, the advanced glycoslation endproduct forms complexeswith one or more binding partners and one member of the complex may belabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by the knownapplicable detection methods.

With reference to the use of an AGE antibody as a binding partner, itwill be seen from the above that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. Where used and for purposes of this description, Ab₁will be referred to as a primary or anti-advanced glycosylationendproduct antibody, and Ab₂ will be referred to as a secondary oranti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

Suitable radioactive elements may be selected from the group consistingof ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, hu 51Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵I, ¹³¹ I, and ¹⁸⁶ Re. In the instance where a radioactive label, such isprepared with one of the above isotopes is used, known currentlyavailable counting procedures may be utilized.

In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectro-photometric, thermometric,amperometric or gasometric techniques known in the art. The enzyme maybe conjugated to the advanced glycosylation endproducts, their bindingpartners or carrier molecules by reaction with bridging molecules suchas carbodiimides, diisocyanates, glutaraldehyde and the like. Also, andin a particular embodiment of the present invention, the enzymesthemselves may be modified into advanced glycosylation endproducts byreaction with sugars as set forth herein.

Many enzymes which can be used in these procedures are known and can beutilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase, hexokinase plus GPDase, RNAse, glucose oxidase plus alkalinephosphatase, NAD oxidoreductase plus luciferase, phosphofructokinaseplus phosphoenol pyruvate carboxylase, aspartate aminotransferase plusphosphoenol pyruvate decarboxylase, and alkaline phosphatase. U.S. Pat.Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by Way ofexample for their disclosure of alternative labeling material andmethods.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The present invention includes assay systems that may be prepared in theform of test kits for the quantitative analysis of the extent of thepresence of advanced glycosylation endproducts. The system or test kitmay comprise a labeled component prepared by one of the radioactiveand/or enzymatic techniques discussed herein, coupling a label to abinding partner to the advanced glycosylation endproduct such as areceptor or ligand as listed herein; and one or more additionalimmunochemical reagents, at least one of which is a free or immobilizedligand, capable either of binding with the labeled component, itsbinding partner, one of the components to be determined or their bindingpartner(s).

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of advanced glycosylation endproducts. In accordancewith the testing techniques discussed above, one class of such kits willcontain at least labeled AGE, or its binding partner as stated above,and directions, of course, depending upon the method selected, e.g.,"competitive", "sandwich", "DASP" and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

For example, a first assay format contemplates a bound receptor to whichare added the ligand and the analyte. The resulting substrate is thenwashed after which detection proceeds by the measurement of the amountof ligand bound to the receptor. A second format employs a bound ligandto which the receptor and the analyte are added. Both of the first twoformats are based on a competitive reaction with the analyte, while athird format comprises a direct binding reaction between the analyte anda bound receptor. In this format a bound receptor-specific carrier orsubstrate is used. The analyte is first added after which the receptoris added, the substrate washed, and the amount of receptor bound to thesubstrate is measured.

More particularly, the present invention includes the followingprotocols within its scope:

I. A method for determining the amount of advanced glycosylationendproducts in an analyte comprising:

A. providing a sample of mesangial cells;

B. inoculating said sample with a known advanced glycosylationendproduct bound to a whole cell; and

C. counting the whole cells of Step B that are bound to and/orinternalized by said sample.

II. A method for determining the amount of advanced glycosylationendproducts in an analyte comprising:

A. providing a sample taken from mesangial cells believed to containreceptors for said advanced glycosylation endproducts;

B. incubating said sample with a radiolabeled complex of an advancedglycosylation endproduct and a protein; and

C. detecting the radioactivity of said sample and counting the number ofreceptors thereon.

III. A method for determining the amount of advanced glycosylationendproducts in an analyte comprising:

A. providing a sample taken from mesangial cells modified to definereceptors for advanced glycosylation endproducts;

B. incubating the sample of Step A with an analyte suspected ofcontaining AGEs for a period of about 30 minutes;

C. applying a quantity of ¹²⁵ I-AGE-BSA to the sample of Step B withremoval of any excess by rinsing; and

D. measuring the amount of said advanced glycosylation endproduct boundto said analyte by detecting the radioactivity of the sample of Step C.

IV. A method for determining the amount of advanced glycosylationendproducts in an analyte comprising:

A. preparing a sample of said analyte by extraction from cellularcomponents and blotting onto a suitable carrier or substrate;

B. incubating the sample after preparation in accordance with Step A, ina blocking buffer;

C. applying a quantity of ¹²⁵ I-AGE BSA to the sample of Step B withremoval of any excess by rinsing; and

D. measuring the amount of said advanced glycosylation endproduct boundto said analyte by detecting the radioactivity of the sample of Step C.

V. A method for determining the amount of advanced glycosylationendproducts in an analyte comprising:

A. preparing a sample of said analyte bound to a substrate;

B. applying to the sample of Step A a quantity of a ligand bearing aknown advanced glycosylation endproduct;

C. applying to the sample of Step B a quantity of an anti-serum toadvanced glycosylation endproducts; and

D. measuring the amount of said advanced glycosylation endproduct boundto said analyte by detecting the quantity of antiserum present on thebound substrate sample of Step C.

Each of the specific protocols set forth above is illustrated in theexamples that follow herein, and reflects the broad latitude of thepresent invention. All of the protocols disclosed herein may be appliedto the qualitative and quantitative determination of advancedglycosylation endproducts and to the concomitant diagnosis andsurveillance of pathologies in which the accretion of advancedglycosylation endproducts is implicated. Such conditions as diabetes andthe conditions associated with aging, such as atherosclerosis and skinwrinkling represent non-limiting examples, and accordingly methods fordiagnosing and monitoring these conditions are included within the scopeof the present invention.

Accordingly, a test kit may be prepared for the demonstration of thepresence and activity of AGEs, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of anadvanced glycoslation endproduct or a specific binding partner thereto,to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of a binding partner to an advanced glycosylationendproduct as described above, or a ligand thereof, generally bound to asolid phase to form an immunosorbent, or in the alternative, bound to asuitable tag, or plural such end products, etc. (or their bindingpartners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may comprise:

(a) a labeled component which has been obtained by coupling the bindingpartner of the advanced glycosylation endproduct to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the advanced glycosylation endproduct and a specificbinding partner thereto.

In the embodiment of the invention wherein antibodies to AGEs are to beused as the binding partner, such antibodies can for example, beproduced and isolated by standard methods including the well knownhybridoma techniques.

The existence of antibodies against advanced glycosylation endproductsmakes possible another method for isolating other AGEs and theirligands. The method takes advantage of an antibody characteristic knownas idiotypy. Each antibody contains a unique region that is specific foran antigen. This region is called the idiotype. Antibodies themselvescontain antigenic determinants; the idiotype of an antibody is anantigenic determinant unique to that molecule. By immunizing an organismwith antibodies, one can raise "anti-antibodies" that recognize them,including antibodies that recognize the idiotype. Antibodies thatrecognize the idiotype of another antibody are called anti-idiotypeantibodies. Some anti-idiotypic antibodies mimic the shape of theoriginal antigen that the antibody recognizes and are said to bear the"internal image" of the antigen. (Kennedy, 1986.) When the antigen is aligand, certain anti-idiotypes that bind to that ligand's receptor.Investigators have identified several of these, including anti-idiotypesthat bind to receptors for insulin, angiotensin II, adenosine I,β-adrenalin, and rat brain nicotine and opiate receptors. (Carlsson andGlad, 1989). Taking advantage of this phenomenon, other ligands may beisolated using anti-idiotypic antibodies. Anti-idiotypes may be used toscreen for molecules binding to the original antigen.

As stated earlier, the mesangial cell receptors and/or its proteins maybe prepared by isolation and purification from MCs known to bear orproduce the receptors and/or its proteins. The cells or active fragmentslikely to participate in receptor protein synthesis or to have receptorprotein associated therewith may be subjected to a series of knownisolation techniques, such as for example elution ofdetergent-solubilized rat MC membrane proteins from an AGE-proteinaffinity matrix, whereupon the present receptor proteins may berecovered. A specific protocol is set forth by way of illustration inthe examples. The present invention naturally contemplates alternatemeans for preparation of the component proteins, including stimulationof receptor producer cells with promoters of receptor synthesis followedby the isolation and recovery of the receptor as indicated above, aswell as chemical synthesis, and the invention is accordingly intended tocover such alternate preparations within its scope.

The present invention also extends to antibodies including polyclonaland monoclonal antibodies, to the receptor proteins that would find usein a variety of diagnostic and therapeutic applications. For example,the antibodies could be used to screen expression libraries to obtainthe gene that encodes either the receptor complex or its componentproteins. Further, those antibodies that neutralize receptor activitycould initially be employed in intact animals to better elucidate thebiological role that the receptor plays. Such antibodies could alsoparticipate in drug screening assays to identify alternative drugs orother agents that may exhibit the same activity as the receptorproteins.

Both polyclonal and monoclonal antibodies to the proteins receptorproteins are contemplated, the latter capable of preparation by wellknown techniques such as the hybridoma technique, utilizing, forexample, fused mouse spleen lymphocytes and myeloma cells. Immortal,antibody-producing cell lines can also be created by techniques otherthan fusion, such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. Specificpolyclonal antibodies can be raised. Naturally, these antibodies aremerely illustrative of antibody preparations that may be made inaccordance with the present invention.

As the receptor proteins appear to play a role in the recognition andremoval of advanced glycosylation endproducts in vivo, the presentinvention contemplates both diagnostic and therapeutic applications forthese agents. Accordingly, the receptor proteins may be prepared for usein a variety of diagnostic methods, set forth in detail hereinafter, andmay be labeled or unlabeled as appropriate. Likewise, the MC receptorproteins may be prepared for administration in various scenarios fortherapeutic purposes, in most instances to assist in reducing theconcentration of AGEs in vivo.

The receptor proteins may be prepared in a therapeutically effectiveconcentration as a pharmaceutical composition with a pharmaceuticallyacceptable carrier. Other compatible pharmaceutical agents may possiblybe included, so that for example certain agents may be simultaneouslycoadministered. Also, the receptor proteins may be associated with orexpressed by a compatible cellular colony, and the resulting cellularmass may then be treated as a therapeutic agent and administered to apatient in accordance with a predetermined protocol. Numeroustherapeutic formulations are possible and the present inventioncontemplates all such variations within its scope. A variety ofadministrative techniques may be utilized, among them topicalapplications as in ointments or on surgical and other topical appliancessuch as, surgical sponges, bandages, gauze pads, and the like. Also,such compositions may be administered by parenteral techniques such assubcutaneous, intravenous and intraperitoneal injections,catheterizations and the like.

Corresponding therapeutic utilities take advantage of the demonstratedactivity of the present receptor complex and/or the component proteinstoward advanced glycosylation endproducts. Thus, to the extent that thein vivo recognition and removal of AGEs serves to treat ailmentsattributable to their presence in an excess concentration, theadministration of the present receptor complex and/or the componentproteins comprises an effective therapeutic method. Such conditions asdiabetic nephropathy, renal failure and the like may be treated and/oraverted by the practice of the therapeutic methods of the presentinvention. Average quantities of the active agent may vary and inparticular should be based upon the recommendations and prescription ofa qualified physician or veterinarian, with an exemplary dosage regimenextending to up to about 25 mg/kg/day.

The present invention also relates to a variety of diagnosticapplications, including methods for the measurement of the presence andamount of advanced glycosylation endproducts in both plants and animals,including humans. The methods comprise assays involving in addition tothe analyte, one or more binding partners of the advanced glycosylationendproducts, and one or more ligands.

The present invention will be better understood from a consideration ofthe following illustrative examples and data. Accordingly, Examples Iand II presented in parent application Ser. No. 453,958 confirm thebasic hypothesis that the in vivo recognition and removal of AGEs isreceptor mediated, and Examples III presents the investigations andexperiments that have resulted in the identification of the mesangialcell-derived AGE receptors of the present invention.

EXAMPLE 1

In this example, the existence of the receptor-mediated clearance systemof advanced glycosylation endproducts that underlies the present assaywas initially explored, in part, by the performance of a competitivephagocytosis assay was conducted with whole monocytes. A full review ofthe details of the experimental procedures involved is presented in U.S.Pat. No. 4,900,747, and reference may be made thereto for such purpose.

In this example, human red blood cells (RBCs) were collected andisolated, and separate quantities were prepared to facilitate theperformance of the assay. Specifically, a quantity of RBCs wereopsonized by incubation with an appropriate antiserum. A furtherquantity was bound to the advanced glycosylation endproduct FFI by acarbodiimide bond, and additional RBCs were separately glycosylated byreactions with glucose, glucose-6-phosphate, xylose and arabinose,respectively. Lastly, AGE-BSA and human monocytes were prepared.Phagocytosis assays proceeded by the incubation of the RBCs with themonocyte cultures followed by fixation of the sample wells and lastlycounting under 40× phase microscopy.

An FFI-RBC half life assay was also conducted with Balb/c mice that wereinoculated with FFI-RBC suspensions labeled with ⁵¹ Cr. The labeledcells were washed at least four times to remove unbound isotope. TwelveBalb/c mice were then injected intravenously with 200 μl RBC suspension.Each sample was administered in three Balb/c mice. At appropriate timeintervals the mice were bled (0.2 ml) and radioactivity levels weremeasured by counting.

RESULTS

Maximum binding of red cells was observed on Day-7 of monocyteincubation in vitro. Maximum binding and endocytosis of FFI-RBC wascomplete within 30-45 minutes while opsonized cells were maximally boundwithin 15 minutes. At the end of one hour incubation of FFI-coupledcoupled RBC's with cultured human monocytes, per cent phagocytosis andphagocytic index were estimated. As shown in FIG. 1, %erythrophagocytosis of FFI-modified red cells (55%) and IgG-coated redcells (70%) were significantly higher than that of control PBS-treatedcells (4%). Similarly the phagocytic index of FFI-treated RBC's wasgreatly elevated (3.4) as compared to normal controls (1.2).

In order to establish the specificity of the interaction of FFI-RBC'swith the human monocytes, competition experiments were carried out inwhich binding and ingestion of red cells was observed in the absence andpresence of AGE-BSA, prepared as described in Methods (Vlassara et al.,supra.). As shown in FIG. 2, the addition of AGE-BSA at concentrationsof 500 μg/ml inhibited the FFI-RBC binding by more than 70% of thecontrol. In contrast, AGE-BSA did not inhibit opsonized or PBS-treatedred cells, even at maximal concentrations (1 mg/ml). These datasuggested that FFI-modified red cells were recognized and boundspecifically by the monocyte AGE-binding site, and consequentlyconfirmed the operability of the present assay.

DISCUSSION

The above tests extend previous observations on the recognition ofadvanced glycosylation endproducts (AGE) by a specificmonocyte/macrophage receptor, and present evidence that such adductsonce attached chemically or formed in vitro on the surface of intacthuman cells can induce cell binding and ingestion by normal humanmonocytes. The experiments establish the development of a competitivereceptor-based assay for AGEs measuring by way of illustration herein,AGE-red cell binding in the presence of large excess of AGE-BSA(Vlassara et al., supra.).

EXAMPLE 2

This example comprises a series of experiments that were initiallyperformed to measure the ability of agents to stimulate phagocytic cellsto stimulate uptake and degradation of endproducts (AGEs), and therebyfurther confirmed the hypothesis that this activity isreceptor-mediated.

Several AGEs were prepared using the same procedure as disclosed inExample 1, above. Accordingly, FFI-HA was prepared as described andquantities were bound to both human and bovine albumin. A water solublecarbodiimide was used to attach the acid moiety of the FFI-HA to anamino group on the protein. After preparation, the conjugate waspurified and then used in vitro to stimulate macrophages, by incubationfor from 4 to 24 hours.

The AGEs that were to be observed for uptake and degradation wereappropriately radiolabeled so that they could be traced. Thereafter, thestimulated macrophages were tested by exposure to the radiolabeled AGEsfollowing exposure to various agents to measure the effect that theseagents had on the ability of the macrophages to take up and degrade thelabeled AGEs. The above procedures conform to the protocol employed byVlassara et al., supra. and confirmed that a competitive assay based ona cellular receptor for AGEs is feasible.

It was also demonstrated that monocyte or macrophage cells can also bestimulated by AGE-carrier molecules which result in cells with enhancedability to bind, internalize and degrade other AGE-molecules.AGE-carrier molecules are made, for example, from the reaction ofglucose or glucose-6-phosphate with albumin. After purification of thereaction product, the AGE-albumin uptake of AGE-macromoleculesdemonstrated as in (A) above. AGE-BSA (prepared from the incubation ofglucose-6-phosphate with albumin for 6-8 weeks) at 0.1 mg/ml demostratesa stimulatory effect on AGE-BSA uptake by human monocytes (FIG. 4, barAA), and shows a slight stimulation at higher concentrations (bars BBand CC). This observation further supports the role of these ligands inconjunction with cellular receptors and point to the application ofthese agents in a competitive AGE assay protocol.

EXAMPLE 3 Materials and Methods Cell culture

Primary cultures of rat MCs were obtained from outgrowths of isolatedrat glomeruli by Dr. M. Ganz (Yale University, N. Haven). In brief, ratswere anesthetized with ether and the kidneys were excised under sterileconditions. After removing the kidney capsule, the kidney cortices wereisolated, minced to a fine paste with a razor blade, and then pressedthrough serial stainless steel sieves (Tyler USA No. 140, 80, and 200).Glomeruli were collected from the top of the 75 micrometer sieve. Thisprocess resulted in >98% pure glomeruli. The glomeruli were thenpelleted and resuspended in DMEM supplemented with 20% FBS, 5 μg/mlbovine insulin, 2 mM L-glutamine, and 40 μg/ml gentamicin. Theglomerular suspensions were plated onto tissue culture flasks andincubated at 37° C. in 5% CO₂. Primary cultures were allowed to grow for3-4 weeks at which time the MCs were confluent. MCs were used betweenthe fourth and ninth passages.

The purity of the rat MC populations was documented. The MCs exhibited auniform straplike appearance and stained positively for Thy 1-1 antigen,myosin and actin. They were sensitive to mitomycin C, a MC toxin, butwere resistant to the aminonucleoside puromycin, an epithelial celltoxin. Fibroblast contamination was excluded by demonstrating theability of the cells to grow in media in which L-valine had beensubstituted for D-valine. In addition, they stained negative for factorVIII and cytokeratin. Over the experimental period they continued tomaintain a uniform stellate appearance.

Human MC were provided by Dr. J. Floege and Dr. K. Resch, (Hannover,FRG). In brief, normal human kidney tissue was obtained from nephrectomyspecimens. Renal cortices were homogenized and glomeruli were isolatedfollowing passage through a series of graded sieves. The glomeruli werethen treated with bacterial collagenase (Worthington BiochemicalCorporation, Freehold, N.J.) at 37° C. for 30 minutes, and afterextensive washing the glomerular remnants were plated onto tissueculture flasks in RPMI 1640, supplemented with 20% FBS, 2 mML-glutamine, 2 mM sodium pyruvate, 5 μg/ml bovine insulin, 5 μg/ml humantransferrin, 1% (v/v) non-essential amino acids, and gentamicin.Cellular outgrowths appeared between days 5-8, and all experiments wereperformed using cells between the fourth and tenth passage.

The purity of the MC population was demonstrated. In brief,immunofluorescent staining demonstrated prominent intracellular stainingfor smooth muscle cell myosin, MHC class I antigen, vimentin, collagenIV, and fibronectin. The cells stained negative for Fc-receptor, MHC IIsurface antigen, cytokeratin and factor VIII, and were able to grow inD-valine substituted medium.

Preparation of ligands

AGE- bovine serum albumin (BSA) and AGE-ribonuclease were made byincubating BSA and bovine ribonuclease (Sigma Chemical Co, St. Louis,Mo.) with 0.5M glucose-6-phosphate (G6P), at 37° C. for 4 to 6 weeks ina 10 mM PBS buffer, pH 7.4, in the presence of protease inhibitors andantibiotics. Unincorporated glucose was removed by dialysis against 1×PBS. The concentration of AGE-BSA was determined and the concentrationof ribonuclease was determined spectrophotometrically.

AGE formed on either BSA or ribonuclease was assessed based oncharacteristic absorption and fluorescence spectra (emission at 450 nm,excitation at 390 nm) and quantitated by a radioreceptor assay usingintact RAW 264.7 cells grown in 96-well plates. According to this assay,AGE-BSA contained approximately 70 AGE U/mg (one unit of AGE is definedas the concentration of unknown agent required to produce 50% inhibitionof standard ¹²⁵ I-AGE-BSA binding) and AGE-ribonuclease contained 62 AGEU/mg.

Borohydride Reduction

To examine the effect of early glycosylation product reduction on ligandbinding, AGE-BSA was incubated with 200 molar excess NaBH₄ (SigmaChemical Co.) for 10 minutes at 4° C., followed by 1 hour at roomtemperature. The reduced AGE-BSA was then dialyzed against 1× PBS andthe protein concentration was determined as above. The chemicallydefined AGE, 2-furoyl-4(5)-2-furanyl-1-H-imidazole (FFI-HA), wassynthesized and linked to BSA with 100 mM water soluble carbodiimide.

Iodination of AGE-BSA

AGE-BSA was iodinated with carrier-free-¹²⁵ I by the IODO-GEN method(Bio-Rad) of Fraker and Speck. Samples were dialyzed against PBSuntil >95% of radioactivity was trichloroacetic acid (TCA)-precipitableand the samples were iodide free.

Preparation of AGE-matrices

6-well plates coated with rat tail collagen, type I, human fibronectin,and polylysine were purchased from Collaborative Research, Inc.(Bedford, Mass.). AGE-matrices were produced by incubating the variousmatrix coated plates in 0.5M G6P, at 37° C. for 2-3 weeks in 10 mM PBSbuffer (pH 7.4), as described for AGE-BSA. Control matrices wereincubated under identical conditions in buffer alone. Followingincubation, the plates were washed extensively with 1× PBS. The amountof adhered collagen I was determined using a hydroxyproline assay, whileadhered fibronectin and laminin were determined by the method of Lowryet al. after dissolving the matrix in 2N NaOH at 37° C. overnight, asdescribed by Jones et al., and by absorbance at 280 nm. In both casessimilar amounts of unmodified or AGE-modified matrix proteins adhered(collagen I, ˜85%; fibronectin, ˜70%; laminin, ˜80% of the plated amountremained attached to the plates). AGE levels in matrix proteins werequantitated by an AGE-specific radioreceptor assay, as described above.AGE-collagen I contained 47 AGE U/mg, AGE-fibronectin 54 AGE U/mg andAGE-laminin, 51 U/mg. Unmodified matrices contained less than 5 AGE U/mgprotein.

Membrane preparation

Rat and human MCs were grown to confluency in 150 mm petri dishes. Cellswere detached from the plates by PBS containing 3% EDTA and proteaseinhibitors (2 mM phenylmethylsulfonyl-fluoride [PMSF], 10 μg/mlaprotinin, 5 ng/ml pepstatin, and 1 mM 1,10-phenanthroline).

Following centrifugation, cells were disrupted with a tight Douncehomogenizer, pestle A, in a solution of PBS, with 10 mM EDTA andprotease inhibitors, as stated above. The nuclear and organelle-enrichedfraction were removed by centrifugation at 13,000× g. Membranes werethen isolated from the supernatant by centrifugation at 100,000× g for 1hour at 4° C. The resulting enriched membrane fraction was solubilizedin PBS, containing 1% triton X-100, and 2 mM PMSF. The proteinconcentration was determined. This material was then used for bindingand ligand blotting studies.

Binding studies

Filter binding studies were performed according to the method ofSchneider et al. and Daniel et al. with minor modifications. 10-20 μg ofMC membrane protein was dot-blotted onto nitrocellulose filters. Thenitrocellulose filters were then cut and each dot was placed in aseparate well of a 24-well plate. Following blocking of the filters for1 hour at 4° C. in PBS containing 1.5% BSA, binding studies wereinitiated by adding various concentrations of radioactive ligand to theindividual wells.

At 2 hours, the nitrocellulose filters were washed 3 times with ice-cold1× PBS, and radioactivity bound was quantitated using a Packard TricarbScintillation counter.

Specific binding was defined as the difference between total binding(radioligand incubated with membrane protein alone), and nonspecificbinding (cells incubated with radiolabelled ligand plus 100-fold excessunlabelled ligand). Scatchard analysis of the data was performed todetermine the binding affinity constant and the receptor number.Competition studies were performed in a similar manner to the bindingstudies, with the exception that the nitrocellulose filters werepreincubated with the competitor for 1 hour before adding theradiolabelled ligand.

Binding studies were also performed on confluent MCs in 6-well plates.The studies were performed in 1 ml of RPMI-1640 at 4° C. following theaddition of various concentrations of radioactive ligand. After 2 hoursof binding, the radioligand-containing medium was removed, and the cellswere washed with ice-cold PBS. The cell monolayer was then disruptedwith 1% triton X-100, and the cell-associated radioactivity wasquantitated. Protein concentration was determined by the method ofBradford. Specific binding was determined in an identical manner to thatdescribed above for the filter binding assay.

Ligand blotting

MC membrane preparations (5 μg aliquots) were electrophoresed on anonreducing SDS-PAGE (10%), and then electroblotted onto anitrocellulose filter. Following blocking for 1 hour in a solution ofPBS containing 1.5% BSA, the nitrocellulose filter was probed with ¹²⁵I- AGE-BSA in the presence of 100-fold excess of either BSA or AGE-BSA.The blot was washed 3 times with I× PBS and exposed to Kodak XAR-5 filmat -80° C.

Uptake and degradation

MC uptake and degradation was performed with a minor modification ofpreviously described procedures. Briefly, MCs were grown to confluencyin 6-well plates in DMEM containing 20% FBS and insulin. MC accumulationof radioactive ligand (AGE-BSA) was assessed by incubating cells withvarious concentrations of ¹²⁵ I-AGE-BSA, in the presence and absence of100-fold excess of unlabelled AGE-BSA, for 4 hours at 37° C. Afterwashing the cells 3 times with ice-cold PBS, the cells were solubilizedin 1% Triton X-100 for 45 minutes at room temperature, and the amount ofcell associated radioactivity was determined. Specific uptake wasdefined by the same criteria as used for the MC binding studies. Proteinconcentration was determined by the method of Bradford. Degradation wasdetermined by measuring TCA-soluble radioactivity in the aspiratedmedium.

Proliferation assays

Rat MCs in DMEM containing 20% FBS were plated at 1×10⁴ cells/well ontoflat-bottom 96-well microtitre plates, which had been pre-coated withdifferent amounts of either AGE-modified or unmodified matrix proteins.24 hours later, the cells were washed with 1× PBS and incubated for anadditional 48 hrs in medium containing 0.3% FBS. The cells were thenlabelled with 2 uCi of (³ H) thymidine (Amersham Corp., ArlingtonHeights, Ill.) for 18-24 hours, following which the supernatants wereaspirated and the cells in each well were harvested onto glass fiberfilters with an automated cell harvester. The amount of (³ H) thymidineincorporated was determined with a Beckman Scintillation Counter.

To confirm thymidine incorporation data, parallel studies were carriedout using an immunocytochemical assay system for detection of DNAsynthesis by measuring bromodeoxyuridine (BrdU) incorporation, while inseparate experiments cells were trypsinized and counted in a Coulterparticle counter. The data obtained by these two additional methods wereconsistent with ³ H-thymidine results (variations between replicatewells deviated no more than 10%).

Fibronectin production

Human MCs in RPMI-1640 medium supplemented with 20% FBS were plated at2×10⁵ cells per well onto 6-well plates which had been coated withglucose-modified or unmodified matrix proteins. After 24 hours, thecells were washed with 1× PBS and incubated in medium containing 0.3%FBS for 48 hours. The cells were then labeled in methionine free mediumfor 3 hours with 200 uCi of (35S) methionine and cysteine (Translabel,ICN).

After labeling, the medium was removed and the cell monolayers werewashed with cold 1× PBS. The monolayers were extracted with 0.5 ml of a1M urea solution containing 1 mM dithiothreitol (DTT), 10 mM Tris-HCL(pH) 7.4), 10 mM EDTA, and 2 mM PMSF.

Fibronectin was then isolated from the medium and matrix byimmunoprecipitation with an IgG purified anti-human fibronectin antibody(Cappel, Malvern, Pa.). Anti-fibronectin antibody was added to thesamples and incubated overnight at 4° C. To insure than any differencesin fibronectin synthesis were not due to different number of cells/well,equal amounts of TCA-precipitable counts were immunoprecipitated fromeach well. The immune complexes were isolated using protein A-Sepharosebeads (Pharmacia).

After washing the protein A-Sepharose beads 3 times in 100 mM Tris HCl(pH 7.4), 0.5% SDS, 0.5% Triton X-100, 2 mM PMSF, and 10 mM EDTA,fibronectin was released by heating at 100° C. for 5 minutes in SDS-PAGEsample buffer, and analyzed by gel electrophoresis and fluorography. Theamount of fibronectin from each sample was quantitated by slicing thefibronectin band from the gel and determining (35S) methionine andcysteine incorporation in a liquid scintillation counter.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. An isolated receptor protein reactive withadvanced glycosylation end products, characterized as follows:(a) it hasa molecular mass from 30 to 50 kD; (b) it is reactive with AGE-BSA,AGE-RNAse, AGE-collagen I and AGE-BSA reduced with NaBH₄ and has abinding affinity of 2.0±0.4×10⁻⁶ M⁻¹ (kD=500 nM); (c) it is non-reactivewith BSA, collagen I, RNAse or chemically synthesized FFI-BSA; and (d)it is present on mesangial cell membranes prior to purification.
 2. Aplurality of isolated receptor proteins reactive with advancedglycosylation endproducts, characterized as follows:(a) at least one ofsaid proteins has a molecular mass from 30 to 50 kD; (b) the pluralityof proteins is derived from mammalian mesangial cells; (c) the proteinsare reactive with AGE-BSA, AGE-RNAse, AGE-collagen I and AGE-BSA reducedwith NaBH₄, having a binding affinity of 2.0±0.4×10⁻⁶ M⁻¹ (kD=500 nM);(d) the proteins are non-reactive with BSA, collagen I, RNAse orchemically synthesized FFI-BSA; and (e) the proteins are present onmesangial cell membranes prior to purification in an amount sufficientto bind about 3.0±0.25×10⁵ AGE-modified protein molecules per mesangialcell.
 3. The protein of claim 1 or the receptor proteins of claim 2labeled with a detectable label.
 4. An isolated receptor proteinreactive with advanced glycosylation end products in accordance withclaim 1 having a molecular mass of about 50 kD.
 5. An isolated receptorprotein reactive with advanced glycosylation end products in accordancewith claim 1 having a molecular mass of about 40 kD.
 6. An isolatedreceptor protein reactive with advanced glycosylation end products inaccordance with claim 1 having a molecular mass of about30-35 kD.